title: Design of an effective mechanism-based inactivator for a zinc protease.
abstract: (R)-2-Benzyl-5-cyano-4-oxopentanoic acid (compound 4) was studied as a mechanism-based inactivator (suicide substrate) for the zinc protease carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1). This compound was designed rationally based on the knowledge of the active site topology and the reported stereospecific proton exchange on ketonic substrate analogue (R)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid [Sugimoto, T. & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751] by CPA. It is suggested that enzymic deprotonation on the C-5 methylene moiety may result in the transient formation of a ketenimine as the key intermediate that partitions between turnover and enzyme inactivation. The enzyme inactivation exhibited pseudo-first-order kinetics, was irreversible, and could be fully prevented in the presence of the reversible inhibitor benzyl-succinate. The inactivation rate constant, kintact, was evaluated to be 0.083 +/- 0.003 min-1 and kcat was measured at 1.78 +/- 0.06 min-1. In turn, a partition ratio of 28 +/- 3 was calculated. The reversible inhibitor constant (Ki) was measured at 1.8 +/- 0.5 microM, indicative of a high affinity for compound 4 shown by CPA; however, Km for the turnover process was determined at 4.93 +/- 0.43 mM. Kinetic analysis and labeling by the radioactive form of the inactivator suggested that the stoichiometry for protein modification by compound 4 approaches a 1:1 ratio.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202.
authors.forename: S
authors.initials: S
authors.lastname: Mobashery
authors.forename: S S
authors.initials: SS
authors.lastname: Ghosh
authors.forename: S Y
authors.initials: SY
authors.lastname: Tamura
authors.forename: E T
authors.initials: ET
authors.lastname: Kaiser
mesh_terms: D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D015195:Drug Design; D007202:Indicators and Reagents; D007700:Kinetics; D007982:Levulinic Acids / Q000138:chemical synthesis* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008962:Models, Theoretical; D011480:Protease Inhibitors / Q000138:chemical synthesis*; D015032:Zinc
chemical_list: D007202:Indicators and Reagents; D007982:Levulinic Acids; D011480:Protease Inhibitors; C062661:2-benzyl-5-cyano-4-oxopentanoic acid; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc